Lettuce cultivar 80-0601013-B

ABSTRACT

A lettuce cultivar, designated 80-0601013-B, is disclosed. The invention relates to the seeds of lettuce cultivar 80-0601013-B, to the plants of lettuce cultivar 80-0601013-B and to methods for producing a lettuce plant by crossing the cultivar 80-0601013-B with itself or another lettuce cultivar. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plants and plant parts produced by those methods. This invention also relates to lettuce cultivars or breeding cultivars and plant parts derived from lettuce cultivar 80-0601013-B, to methods for producing other lettuce cultivars, lines or plant parts derived from lettuce cultivar 80-0601013-B and to the lettuce plants, varieties, and their parts derived from the use of those methods. The invention further relates to hybrid lettuce seeds, plants, and plant parts produced by crossing cultivar 80-0601013-B with another lettuce cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive lollo verdelettuce (Lactuca sativa L.) variety designated 80-0601013-B. Allpublications cited in this application are herein incorporated byreference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety or hybridan improved combination of desirable traits from the parental germplasm.These important traits may include increased head size and weight,higher seed yield, improved color, resistance to diseases and insects,tolerance to drought and heat, and better agronomic quality.

Practically speaking, all cultivated forms of lettuce belong to thehighly polymorphic species Lactuca sativa that is grown for its ediblehead and leaves. As a crop, lettuce is grown commercially whereverenvironmental conditions permit the production of an economically viableyield. Lettuce is the World's most popular salad. In the United States,the principal growing regions are California and Arizona which produceapproximately 329,700 acres out of a total annual acreage of more than333,300 acres (USDA, 2005). Fresh lettuce is available in the UnitedStates year-round although the greatest supply is from May throughOctober. For planting purposes, the lettuce season is typically dividedinto three categories, early, mid and late, with the coastal areasplanting from January to August, and the desert regions planting fromAugust to December. Fresh lettuce is consumed nearly exclusively asfresh, raw product and occasionally as a cooked vegetable.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae)family. Lettuce is related to chicory, sunflower, aster, dandelion,artichoke and chrysanthemum. Sativa is one of about 300 species in thegenus Lactuca. There are seven different morphological types of lettuce.The crisphead group includes the iceberg and batavian types. Iceberglettuce has a large, firm head with a crisp texture and a white orcreamy yellow interior. The batavian lettuce predates the iceberg typeand has a smaller and less firm head. The butterhead group has a small,soft head with an almost oily texture. The romaine, also known as coslettuce, has elongated upright leaves forming a loose, loaf-shaped headand the outer leaves are usually dark green. Leaf lettuce comes in manyvarieties, none of which form a head, and include the green oak leafvariety. The next three types are seldom seen in the United States:Latin lettuce looks like a cross between romaine and butterhead; stemlettuce has long, narrow leaves and thick, edible stems; and oilseedlettuce is a type grown for its large seeds that are pressed to obtainoil.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard, theoverall value of the advanced breeding lines, and the number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for at least three years. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits areused as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from ten to twenty years from the time thefirst cross or selection is made. Therefore, development of newcultivars is a time-consuming process that requires precise forwardplanning, efficient use of resources, and a minimum of changes indirection.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of lettuce plant breeding is to develop new, unique andsuperior lettuce cultivars. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations. The breeder has no direct control at the cellularlevel. Therefore, two breeders will never develop the same line, or evenvery similar lines, having the same lettuce traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under different geographical,climatic and soil conditions, and further selections are then made,during and at the end of the growing season. The cultivars that aredeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop superior lettuce cultivars.

The development of commercial lettuce cultivars requires the developmentof lettuce varieties, the crossing of these varieties, and theevaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichcultivars are developed by selfing and selection of desired phenotypes.The new cultivars are crossed with other varieties and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, (Molecular Linkage Map ofSoybean (Glycine max) p 6.131-6.138 in S. J. O'Brien (ed) Genetic Maps:Locus Maps of Complex Genomes, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1993)) developed a molecular genetic linkage mapthat consisted of 25 linkage groups with about 365 RFLP, 11 RAPD, threeclassical markers and four isozyme loci. See also, Shoemaker, R. C.,RFLP Map of Soybean, p 299-309, in Phillips, R. L. and Vasil, I. K.,eds. DNA-Based Markers in Plants, Kluwer Academic Press, Dordrecht, theNetherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. (Diwan, N. and Cregan, P.B., Theor. Appl. Genet. 95:22-225, 1997.) SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding is another method of introducing new traits intolettuce varieties. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation (such as X-rays, Gamma rays,neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens(such as base analogs like 5-bromo-uracil), antibiotics, alkylatingagents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Principles of Cultivar Development byFehr, Macmillan Publishing Company, 1993.

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan et al., Theor. Appl. Genet., 77:889-892, 1989.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Principles of Plant Breeding John Wiley and Son, pp.115-161,1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987; “Carrots and Related Vegetable Umbelliferae”, Rubatzky, V. E., etal., 1999).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Lettuce in general and leaf lettuce in particular is an important andvaluable vegetable crop. Thus, a continuing goal of lettuce plantbreeders is to develop stable, high yielding lettuce cultivars that areagronomically sound. To accomplish this goal, the lettuce breeder mustselect and develop lettuce plants with traits that result in superiorcultivars.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools, and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the invention, there is provided a novel lettuce cultivardesignated 80-0601013-B. This invention thus relates to the seeds oflettuce cultivar 80-0601013-B, to the plants of lettuce cultivar80-0601013-B and to methods for producing a lettuce plant produced bycrossing the lettuce cultivar 80-0601013-B with itself or anotherlettuce plant, to methods for producing a lettuce plant containing inits genetic material one or more transgenes and to the transgeniclettuce plants produced by that method. This invention also relates tomethods for producing other lettuce cultivars derived from lettucecultivar 80-0601013-B and to the lettuce cultivar derived by the use ofthose methods. This invention further relates to hybrid lettuce seedsand plants produced by crossing lettuce cultivar 80-0601013-B withanother lettuce variety.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of lettuce cultivar 80-0601013-B. The tissueculture will preferably be capable of regenerating plants havingessentially all of the physiological and morphological characteristicsof the foregoing lettuce plant, and of regenerating plants havingsubstantially the same genotype as the foregoing lettuce plant.Preferably, the regenerable cells in such tissue cultures will becallus, protoplasts, meristematic cells, cotyledons, hypocotyl, leaves,pollen, embryos, roots, root tips, anthers, pistils, flowers and seeds.Still further, the present invention provides lettuce plants regeneratedfrom the tissue cultures of the invention.

Another aspect of the invention is to provide methods for producingother lettuce plants derived from lettuce cultivar 80-0601013-B. Lettucecultivars derived by the use of those methods are also part of theinvention.

The invention also relates to methods for producing a lettuce plantcontaining in its genetic material one or more transgenes and to thetransgenic lettuce plant produced by those methods.

In another aspect, the present invention provides for single geneconverted plants of 80-0601013-B. The single transferred gene maypreferably be a dominant or recessive allele. Preferably, the singletransferred gene will confer such traits as male sterility, herbicideresistance, insect resistance, modified fatty acid metabolism, modifiedcarbohydrate metabolism, resistance for bacterial, fungal, or viraldisease, male fertility, enhanced nutritional quality and industrialusage. The single gene may be a naturally occurring lettuce gene or atransgene introduced through genetic engineering techniques.

The invention further provides methods for developing lettuce plants ina lettuce plant breeding program using plant breeding techniquesincluding recurrent selection, backcrossing, pedigree breeding,restriction fragment length polymorphism enhanced selection, geneticmarker enhanced selection and transformation. Seeds, lettuce plants, andparts thereof, produced by such breeding methods are also part of theinvention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference bystudy of the following descriptions.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative forms of a gene,all of which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Bolting. The premature development of a flowering stalk, and subsequentseed, before a plant produces a food crop. Bolting is typically causedby late planting when temperatures are low enough to cause vernalizationof the plants.

Bremia lactucae. A common fungus that causes downy mildew in lettuce incooler growing regions.

Core length. Length of the internal lettuce stem measured from the baseof the cut and trimmed head to the tip of the stem.

Cotyledon. One of the first leaves of the embryo of a seed plant;typically one or more in monocotyledons, two in dicotyledons and two ormore in gymnosperms.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date.

Head diameter. Diameter of the cut and trimmed head, sliced vertically,and measured at the widest point perpendicular to the stem.

Head height. Height of the cut and trimmed head, sliced vertically, andmeasured from the base of the cut stem to the cap leaf.

Head weight. Weight of saleable lettuce head, cut and trimmed to marketspecifications.

Maturity date. Maturity refers to the stage when the plants are of fullsize or optimum weight, in marketable form or shape to be of commercialor economic value.

Quantitative Trait Loci. Quantitative Trait Loci (QTL) refers to geneticloci that control to some degree, numerically representable traits thatare usually continuously distributed.

Ratio of head height/diameter. Head height divided by the head diameteris an indication of the head shape; <1 is flattened, 1=round, and >1 ispointed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system, The chart may bepurchased from Royal Horticulture Society Enterprise Ltd RHS Garden;Wisley, Woking; Surrey GU236QB, UK.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing or via genetic engineering wherein essentially all of thedesired morphological and physiological characteristics of a line arerecovered in addition to the single gene transferred into the line viathe backcrossing technique or via genetic engineering.

DETAILED DESCRIPTION OF THE INVENTION

Lettuce cultivar 80-0601013-B is a mildew resistant, lollo verde lettucesuitable for baby-leaf, sandwich leaf, and full head salad production.It is the result of numerous generations of plant selections chosen fortheir dark green, thick leaves. Lettuce cultivar 80-0601013-B is widelyadapted, but is particularly cultivated in California.

Initial trials in Europe generated a high level of interest in thislettuce variety due to its high volume, dark green color and Bremialactucae resistance. While lettuce cultivar 80-0601013-B is similar tothe commercial lettuce cultivars ‘Intrigue’ and ‘Squadron’, lettucecultivar 80-0601013-B has a smaller leaf with a more frilly leaf margin.

Some of the criteria used for selection in various generations include:size, shape, leaf thickness and leaf shape, color, disease resistanceand weight.

The cultivar has shown uniformity and stability for the traits, withinthe limits of environmental influence for the traits. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in cultivar 80-0601013-B.

Lettuce cultivar 80-0601013-B has the following morphologic and othercharacteristics (based primarily on data collected at Gilroy,California).

TABLE 1 VARIETY DESCRIPTION INFORMATION Plant Type: Cutting Leaf Seed:Color: Black Light dormancy: Light not required Heat dormancy: Notsusceptible Cotyledons: Shape: Intermediate Shape of fourth leaf:Elongated Length/width index of 4^(th) leaf (4w × 10): 20 Apex:Crenate/Gnawed Base: Crenate/Gnawed Undulation: Medium Green color: RHS143C (Light green) Anthocyanin distribution: Absent Rolling: PresentCupping: Uncupped Reflexing: Apical margin Mature Leaves: Margin:Incision depth: Deep Indentation: Deeply dentate Undulation of theapical margin: Strong Green color (at harvest maturity): RHS 143C(Medium-green) Anthocyanin distribution: Absent Size: Small Glossiness:Moderate Blistering: Absent to slight Thickness: Thick Trichomes:Present Plant (at market stage): Spread of frame leaves: 24 cm Headshape: Non-heading Head size class: Small Head weight: 360 g Headfirmness: Loose Butt: Shape: Rounded Midrib: Flattened Core: Diameter atbase of head: 2.1 cm Core height from base of head to apex: 4.0 cmBolting: Days to seed stalk emergence from first water date (summerconditions): 42 Bolting class: Rapid Height of mature seed stalk: 89 cmSpread of bolter plant: 29 cm Bolter leaves: Curved Bolter leaf margin:Dentate Bolter leaf color: RHS 146A (Medium green) Bolter habit:Terminal inflorescence: Present Lateral shoots: Present Basal sideshoots: Absent

Disease/Pest Resistance: Resistant to Bremia lactucae California racesCAI-V1 and VIII as well as European isolates BL1-23 and 25.

Lettuce cultivar 80-0601013-B is similar to the commercial lettucecultivar ‘Intrigue’, however there are a number of differences. Forexample, lettuce cultivar 80-0601013-B has a leaf margin with a verystrong undulation, while lettuce cultivar ‘Intrigue’ has a leaf marginwith only strong undulation. Lettuce cultivar 80-0601013-B has a leafcolor of RHS146B (Medium green), while lettuce cultivar ‘Intrigue’ has aleaf color of RHS143C (Medium green). Additionally, lettuce cultivar80-0601013-B has a small head size, while lettuce cultivar ‘Intrigue’has a medium head size.

This invention is also directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant, wherein the first parent lettuce plant or second parent lettuceplant is the lettuce plant from cultivar 80-0601013-B. Further, both thefirst parent lettuce plant and second parent lettuce plant may be fromcultivar 80-0601013-B. Therefore, any methods using lettuce cultivar80-0601013-B are part of this invention: selfing, backcrosses, hybridbreeding, and crosses to populations. Any plants produced using lettucecultivar 80-0601013-B as at least one parent are within the scope ofthis invention.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably, expression vectors are introduced into planttissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformed plants obtained with the protoplasm of the invention areintended to be within the scope of this invention.

FURTHER EMBODIMENTS OF THE INVENTION

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed line.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed lettuce plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the lettuce plant(s).

Expression Vectors for Lettuce Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalswhich confers resistance to kanamycin (Fraley et al., Proc. Natl. Acad.Sci. U.S.A., 80:4803 (1983)). Another commonly used selectable markergene is the hygromycin phosphotransferase gene which confers resistanceto the antibiotic hygromycin (Vanden Elzen et al., Plant Mol. Biol.,5:299 (1985)).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant (Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet, 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol.7:171 (1986)). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil (Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988)).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase (Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643(1990)).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include α-glucuronidase (GUS), α-galactosidase,luciferase and chloramphenicol, acetyltransferase (Jefferson, R. A.,Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989),Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84:131 (1987), DeBlock etal., EMBO J. 3:1681 (1984)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available (Molecular Probes publication2908, IMAGENE GREEN, p. 1-4 (1993) and Naleway et al., J. Cell Biol.115:15a (1991)). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells (Chalfie et al., Science 263:802 (1994)). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Lettuce Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inlettuce. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in lettuce. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen. Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression inlettuce or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in lettuce.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); PEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal2 (3):291-300 (1992)). The ALS promoter, Xba1/NcoI fragment 5′ to the Brassicanapus ALS3 structural gene (or a nucleotide sequence similarity to saidXba1/NcoI fragment), represents a particularly useful constitutivepromoter. See PCT application WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin lettuce. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in lettuce. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zml3(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is lettuce. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology, CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes That Confer Resistance to Pests or Disease and That Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with a clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT application US93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO 95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT application WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. SeeTaylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

S. A lettuce mosaic potyvirus (LMV) coat protein gene introduced intoLactuca sativa in order to increase its resistance to LMV infection. SeeDinant et al., Molecular Breeding. 1997, 3: 1, 75-86.

2. Genes That Confer Resistance to an Herbicide:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin-acetyl transferase PAT bar genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession number 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. See also Umaballava-Mobapathie in TransgenicResearch. 1999, 8: 1, 33-44 that discloses Lactuca sativa resistant toglufosinate. European patent application No. 0 333 033 to Kumada et al.,and U.S. Pat. No. 4,975,374 to Goodman et al., disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori et al., Mol. Gen.Genet. 246:419, 1995. Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., PlantPhysiol., 106:17, 1994), genes for glutathione reductase and superoxidedismutase (Aono et al., Plant Cell Physiol. 36:1687, 1995), and genesfor various phosphotransferases (Datta et al., Plant Mol. Biol. 20:619,1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837;5,767,373; and international publication WO 01/12825.

3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

A. Increased iron content of the lettuce, for example by transforming aplant with a soybean ferritin gene as described in Goto et al., ActaHordiculturae. 2000, 521,101-109.

B. Decreased nitrate content of leaves, for example by transforming alettuce with a gene coding for a nitrate reductase. See for exampleCurtis et al., Plant Cell Report. 1999,18: 11, 889-896.

C. Increased sweetness of the lettuce by transferring a gene coding formonellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia et al., Biotechnology. 1992,10: 561-564.

D. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89:2625 (1992).

E. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

4. Genes that Control Male-Sterility

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See international publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See internationalpublications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611-622, 1992).

Methods for Lettuce Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985), Curtis et al., Journal ofExperimental Botany. 1994, 45: 279, 1441-1449, Torres et al., Plant cellTissue and Organic Culture. 1993, 34: 3, 279-285, Dinant et al.,Molecular Breeding. 1997, 3: 1, 75-86. A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Several methods of plant transformation collectively referred to asdirect gene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 to 4 μm.The expression vector is introduced into plant tissues with a biolisticdevice that accelerates the microprojectiles to speeds of 300 to 600 m/swhich is sufficient to penetrate plant cell walls and membranes.Russell, D. R., et al. Pl. Cell. Rep. 12(3, Jan.), 165-169 (1993),Aragao, F. J. L., et al. Plant Mol. Biol. 20(2, Oct.), 357-359 (1992),Aragao, F. J. L., et al. PL. Cell. Rep. 12(9, July), 483-490 (1993).Aragao, Theor. Appl. Genet. 93: 142-150 (1996), Kim, J.; Minamikawa, T.Plant Science 117: 131-138 (1996), Sanford et al., Part. Sci. Technol.5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al.,Bio/Technology 6:559-563 (1988), Sanford, J. C., Physiol Plant 7:206(1990), Klein et al., Biotechnology 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M.; Kuhne, T. Biologia Plantarum 40(4): 507-514(1997/98), Donn et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin etal., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol.24:51-61 (1994). See also Chupean et al., Biotechnology. 1989, 7: 5,503-508.

Following transformation of lettuce target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossed,with another (non-transformed or transformed) line, in order to producea new transgenic lettuce line. Alternatively, a genetic trait which hasbeen engineered into a particular lettuce cultivar using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Single-Gene Conversions

When the terms lettuce plant, cultivar or lettuce line are used in thecontext of the present invention, this also includes any single geneconversions of that line. The term “single gene converted plant” as usedherein refers to those lettuce plants which are developed by a plantbreeding technique called backcrossing wherein essentially all of thedesired morphological and physiological characteristics of a cultivarare recovered in addition to the single gene transferred into the linevia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into theline. The term “backcrossing” as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental lettuce plantsfor that line, backcrossing 1, 2, 3, 4, 5, 6, 7, 8 or more times to therecurrent parent. The parental lettuce plant which contributes the genefor the desired characteristic is termed the nonrecurrent or donorparent. This terminology refers to the fact that the nonrecurrent parentis used one time in the backcross protocol and therefore does not recur.The parental lettuce plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol (Poehlman &Sleper, 1994; Fehr, 1987). In a typical backcross protocol, the originalcultivar of interest (recurrent parent) is crossed to a second line(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until alettuce plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalline. To accomplish this, a single gene of the recurrent cultivar ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original line. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new line but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, examples of these traits include but are not limited to,male sterility, modified fatty acid metabolism, modified carbohydratemetabolism, herbicide resistance, resistance for bacterial, fungal, orviral disease, insect resistance, enhanced nutritional quality,industrial usage, yield stability and yield enhancement. These genes aregenerally inherited through the nucleus. Several of these single genetraits are described in U.S. Pat. Nos. 5,777,196, 5,948,957 and5,969,212, the disclosures of which are specifically hereby incorporatedby reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of lettuce andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng et al., HortScience. 1992, 27: 9,1030-1032 Teng et al., HortScience. 1993, 28: 6, 669-1671, Zhang et al.,Journal of Genetics and Breeding. 1992, 46: 3, 287-290, Webb et al.,Plant Cell Tissue and Organ Culture. 1994, 38: 1, 77-79, Curtis et al.,Journal of Experimental Botany. 1994, 45: 279, 1441-1449, Nagata et al.,Journal for the American Society for Horticultural Science. 2000,125: 6,669-672, and Ibrahim et al., Plant Cell, Tissue and Organ Culture.(1992), 28(2): 139-145. It is clear from the literature that the stateof the art is such that these methods of obtaining plants are routinelyused and have a very high rate of success. Thus, another aspect of thisinvention is to provide cells which upon growth and differentiationproduce lettuce plants having the physiological and morphologicalcharacteristics of variety 80-0601013-B.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

Additional Breeding Methods

This invention also is directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant wherein the first or second parent lettuce plant is a lettuceplant of cultivar 80-0601013-B. Further, both first and second parentlettuce plants can come from lettuce cultivar 80-0601013-B. Thus, anysuch methods using lettuce cultivar 80-0601013-B are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using lettuce cultivar80-0601013-B as at least one parent are within the scope of thisinvention, including those developed from cultivars derived from lettucecultivar 80-0601013-B. Advantageously, this lettuce cultivar could beused in crosses with other, different, lettuce plants to produce thefirst generation (F₁) lettuce hybrid seeds and plants with superiorcharacteristics. The cultivar of the invention can also be used fortransformation where exogenous genes are introduced and expressed by thecultivar of the invention. Genetic variants created either throughtraditional breeding methods using lettuce cultivar 80-0601013-B orthrough transformation of cultivar 80-0601013-B by any of a number ofprotocols known to those of skill in the art are intended to be withinthe scope of this invention.

The following describes breeding methods that may be used with lettucecultivar 80-0601013-B in the development of further lettuce plants. Onesuch embodiment is a method for developing cultivar 80-0601013-B progenylettuce plants in a lettuce plant breeding program comprising: obtainingthe lettuce plant, or a part thereof, of cultivar 80-0601013-B,utilizing said plant or plant part as a source of breeding material, andselecting a lettuce cultivar 80-0601013-B progeny plant with molecularmarkers in common with cultivar 80-0601013-B and/or with morphologicaland/or physiological characteristics selected from the characteristicslisted in Table 1. Breeding steps that may be used in the lettuce plantbreeding program include pedigree breeding, backcrossing, mutationbreeding, and recurrent selection. In conjunction with these steps,techniques such as RFLP-enhanced selection, genetic marker enhancedselection (for example SSR markers) and the making of double haploidsmay be utilized.

Another method involves producing a population of lettuce cultivar80-0601013-B progeny lettuce plants, comprising crossing cultivar80-0601013-B with another lettuce plant, thereby producing a populationof lettuce plants, which, on average, derive 50% of their alleles fromlettuce cultivar 80-0601013-B. A plant of this population may beselected and repeatedly selfed or sibbed with a lettuce cultivarresulting from these successive filial generations. One embodiment ofthis invention is the lettuce cultivar produced by this method and thathas obtained at least 50% of its alleles from lettuce cultivar80-0601013-B.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus the invention includes lettucecultivar 80-0601013-B progeny lettuce plants comprising a combination ofat least two cultivar 80-0601013-B traits selected from the groupconsisting of those listed in Table 1 or the cultivar 80-0601013-Bcombination of traits listed in the Summary of the Invention, so thatsaid progeny lettuce plant is not significantly different for saidtraits than lettuce cultivar 80-0601013-B as determined at the 5%significance level when grown in the same environmental conditions.Using techniques described herein, molecular markers may be used toidentify said progeny plant as a lettuce cultivar 80-0601013-B progenyplant. Mean trait values may be used to determine whether traitdifferences are significant, and preferably the traits are measured onplants grown under the same environmental conditions. Once such avariety is developed its value is substantial since it is important toadvance the germplasm base as a whole in order to maintain or improvetraits such as yield, disease resistance, pest resistance, and plantperformance in extreme environmental conditions.

Progeny of lettuce cultivar 80-0601013-B may also be characterizedthrough their filial relationship with lettuce cultivar 80-0601013-B, asfor example, being within a certain number of breeding crosses oflettuce cultivar 80-0601013-B. A breeding cross is a cross made tointroduce new genetics into the progeny, and is distinguished from across, such as a self or a sib cross, made to select among existinggenetic alleles. The lower the number of breeding crosses in thepedigree, the closer the relationship between lettuce cultivar80-0601013-B and its progeny. For example, progeny produced by themethods described herein may be within 1, 2, 3, 4 or 5 breeding crossesof lettuce cultivar 80-0601013-B.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which lettuce plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,seeds, stems and the like.

Tables

Table 2 below compares some of the characteristics of leaf lettucecultivar 80-0601013-B with two commercial leaf lettuce cultivars. Column1 lists the characteristics, column 2 shows the characteristics forlettuce cultivar 80-0601013-B, column 3 shows the characteristics forlettuce cultivar ‘Intrigue’ and column 4 shows the characteristics forlettuce cultivar ‘Squadron’.

TABLE 2 Cultivars Characteristic 80-0601013-B ‘Intrigue’ ‘Squadron’ SeedColor Black White Black Length/Width index of 4^(th) 20 23 20 leaf (L/W× 10) Basal margin Crenate/ Entire Crenate/ Gnawed Gnawed Anthocyanincupping Uncupped Slight Slight Leaf margin incision depth Deep ModerateDeep Leaf margin indentation Deeply dentate Crenate Deeply dentateAnthocyanin size Small Small Medium Glossiness Moderate Moderate GlossySpread of frame leaves 24 cm 27 cm 27 cm Head size Small Small MediumHead weight 360 g 377 g 501 g Diameter of core at base of 2.1 cm 2.5 cm2.5 cm head Core height from base of 4.0 cm 3.5 cm 3.4 cm head to apexNumber of days from first 42 48 57 water date to seed stalk emergenceBolting class Rapid Rapid Medium Mature seed stalk height 89 cm 125 cm92 cm

Deposit Information

A deposit of the Shamrock Seed Company, Inc. proprietary lettucecultivar designated 80-0601013-B disclosed above and recited in theappended claims has been made with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110. The date ofdeposit was Sep. 28, 2007. The deposit of 2,500 seeds was taken from thesame deposit maintained by Shamrock Seed Company, Inc. since prior tothe filing date of this application. All restrictions upon the deposithave been removed, and the deposit is intended to meet all of therequirements of 37 C.F.R. 1.801-1.809. The ATCC accession number isPTA-8672. The deposit will be maintained in the depository for a periodof 30 years. or 5 years after the last request, or for the effectivelife of the patent, whichever is longer, and will be replaced asnecessary during that period.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

1. A seed of lettuce cultivar designated 80-0601013-B, wherein arepresentative sample of seed of said cultivar was deposited under ATCCAccession No. PTA-8672.
 2. A lettuce plant, or a part thereof, producedby growing the seed of claim
 1. 3. A tissue culture of cells producedfrom the plant of claim 2, wherein said cells of the tissue culture areproduced from a plant part selected from the group consisting of embryo,meristematic cell, leaf, cotyledon, hypocotyl, stem, root, root tip,pistil, anther, flower, seed and pollen.
 4. A protoplast produced fromthe plant of claim
 2. 5. A protoplast produced from the tissue cultureof claim
 3. 6. A lettuce plant regenerated from the tissue culture ofclaim 3, wherein the plant has all of the morphological andphysiological characteristics of cultivar 80-0601013-B, wherein arepresentative sample of seed was deposited under ATCC Accession No.PTA-8672.
 7. A method for producing a hybrid lettuce seed, wherein themethod comprises crossing the plant of claim 2 with a different lettuceplant and harvesting the resultant F₁ hybrid lettuce seed.
 8. A hybridlettuce seed produced by the method of claim
 7. 9. A hybrid lettuceplant, or a part thereof, produced by growing said hybrid seed of claim8.
 10. A method of producing a male sterile lettuce plant wherein themethod comprises transforming the lettuce plant of claim 2 with anucleic acid molecule that confers male sterility.
 11. A male sterilelettuce plant produced by the method of claim
 10. 12. A method forproducing an herbicide resistant lettuce plant wherein the methodcomprises transforming the lettuce plant of claim 2 with a transgene,wherein the transgene confers resistance to an herbicide selected fromthe group consisting of imidazolinone, sulfonylurea, glyphosate,glufosinate, L-phosphinothricin, triazine and benzonitrile.
 13. Anherbicide resistant lettuce plant produced by the method of claim 12.14. A method of producing an insect resistant lettuce plant wherein themethod comprises transforming the lettuce plant of claim 2 with atransgene that confers insect resistance.
 15. An insect resistantlettuce plant produced by the method of claim
 14. 16. The lettuce plantof claim 15 wherein the transgene encodes a Bacillus thuringiensisendotoxin.
 17. A method of producing a disease resistant lettuce plantwherein the method comprises transforming the lettuce plant of claim 2with a transgene that confers disease resistance.
 18. A diseaseresistant lettuce plant produced by the method of claim
 17. 19. A methodof producing a lettuce plant with a value-added trait, wherein themethod comprises transforming the lettuce plant of claim 2 with atransgene encoding a protein selected from the group consisting of aferritin, a nitrate reductase, and a monellin.
 20. A lettuce plant witha value-added trait produced by the method of claim
 19. 21. A lettuceplant, or a part thereof, having all of the physiological andmorphological characteristics of lettuce cultivar 80-0601013-B, whereina representative sample of seed of the cultivar was deposited under ATCCAccession No. PTA-8672.
 22. A method of introducing a desired trait intolettuce cultivar 80-0601013-B wherein the method comprises: a) crossinga 80-0601013-B plant grown from 80-0601013-B seed, wherein arepresentative sample of seed was deposited under ATCC Accession No.PTA-8672, with a plant of another lettuce cultivar that comprises adesired trait to produce F₁ progeny plants, wherein the desired trait isselected from the group consisting of male sterility, herbicideresistance, insect resistance, and resistance to bacterial disease,fungal disease, or viral disease; b) selecting one or more progenyplants that have the desired trait to produce selected progeny plants;c) crossing the selected progeny plants with the 80-0601013-B plants toproduce backcross progeny plants; d) selecting for backcross progenyplants that have the desired trait and all of the physiological andmorphological characteristics of lettuce cultivar 80-0601013-B listed inTable 1 to produce selected backcross progeny plants; and e) repeatingsteps (c) and (d) three or more times in succession to produce selectedfourth or higher backcross progeny plants that comprise the desiredtrait and all of the physiological and morphological characteristics oflettuce cultivar 80-0601013-B listed in Table
 1. 23. A lettuce plantproduced by the method of claim 22, wherein the plant has the desiredtrait and all of the physiological and morphological characteristics oflettuce cultivar 80-0601013-B listed in Table
 1. 24. The lettuce plantof claim 23, wherein the desired trait is herbicide resistance and theresistance is conferred to an herbicide selected from the groupconsisting of imidazolinone, sulfonylurea, glyphosate, glufosinate,L-phosphinothricin, triazine and benzonitrile.
 25. The lettuce plant ofclaim 23, wherein the desired trait is insect resistance and the insectresistance is conferred by a transgene encoding a Bacillus thuringiensisendotoxin.
 26. The lettuce plant of claim 23, wherein the desired traitis male sterility and the trait is conferred by a nucleic acid molecule.